NMR studies of folded and unfolded proteins: method developments and biological insight

Nur Oktaviani

NMR studies of folded and unfolded proteins: method developments and biological insight

Nur Oktaviani

Moleculaire Dynamica

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Samenvatting

Eiwitten zijn veelvoorkomend in alle levende organismen, en zijn verantwoordelijk voor verschillende biologische functies. NMR spectroscopie is de aangewezen techniek om een ruime hoeveelheid informatie in te winnen omtrent structuur, flexibiliteit en elektrostatische interacties in eiwitten in oplossing, alsmede eiwit-eiwit interacties. Deze informatie is van groot belang voor een overzichtelijk begrip van biologische mechanismen in atomair detail.
In dit proefschrift is biologisch relevant inzicht verkregen over een gevouwen eiwit (photoactive yellow protein (PYP)), alsmede over oplosbaar ontvouwen amyloïde-beta(Abeta peptiden, met behulp van NMR spektroskopie. Een uitgebreide bepaling van alle pKa-constanten voor de zijketens van PYP werd uitgevoerd om inzicht te verschaffen in de elektrostatisch interacties in dit eiwit, en in het bijzonder in het actieve centrum. Daarnaast werd de conformatie van oplosbare covalente Abeta dimeren bestudeerd met NMR spectroscopie en verscheidene andere biofysische methoden, ten einde inzicht te verkrijgen in de bouwstenen van voor de cel giftige aggregaten, die verantwoordelijk worden gehouden voor de ziekte van Alzheimer.

Twee nieuwe methodieken werden ontwikkeld voor dit onderzoek:
(1) Een nieuwe aanpak om de pKa-constanten van tyrosineresiduen te bepalen in gevouwen eiwitten
(2) Een eenvoudige manier om NMR-metingen aan natief ontvouwen eiwitten te versnellen door gebruik te maken van opgeloste paramagnetische complexen
Het onderzoek gepresenteerd in dit proefschrift kan hopelijk van nut zijn voor andere onderzoekers in de NMR-gemeenschap.

Originele taal-2	English
Kwalificatie	Doctor of Philosophy
Toekennende instantie	Rijksuniversiteit Groningen
Begeleider(s)/adviseur	Marrink, Siewert, Supervisor Mulder, Franciscus, Co-supervisor
Datum van toekenning	11-apr.-2014
Plaats van publicatie	[S.l.]
Uitgever	s.n.
Gedrukte ISBN's	978-90-367-6939-6
Elektronische ISBN's	978-90-367-6940-2
Status	Published - 2014

Toegang tot document

Title and contentFinal publisher's version, 311 KB
Chapter 1Final publisher's version, 2,42 MB
Chapter 2Final publisher's version, 1,77 MB
Chapter 3Final publisher's version, 6 MB
Chapter 4Final publisher's version, 2 MB
Chapter 5Final publisher's version, 1,92 MB
Chapter 6Final publisher's version, 540 KB
Summary and future perspectivesFinal publisher's version, 150 KB
Abstract SamenvattingFinal publisher's version, 167 KB
List of publications, AcknowledgementsFinal publisher's version, 214 KB
Complete dissertationFinal publisher's version, 15,4 MB

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title = "NMR studies of folded and unfolded proteins: method developments and biological insight ",

abstract = "Proteins are ubiquitous in every living organism, and perform various biological functions. NMR spectroscopy is the most suitable technique to obtain comprehensive information about structure, dynamics, and electrostatics in proteins in solution, as well as protein-protein interactions. This information is essential for a comprehensive understanding of biological mechanisms at atomic detail. In this dissertation, biological insight about a folded protein (i.e. photoactive yellow protein (PYP)) as well as soluble unfolded amyloid-beta (Abeta peptides was obtained using NMR spectroscopy. For PYP, a comprehensive sidechain pKa determination was performed to obtain an understanding of the electrostatic interactions between charged groups, particularly within the active site. Second, the conformations of soluble covalent Abeta dimers were investigated using NMR spectroscopy along with various other biophysical techniques, to obtain detailed insight into the basic building blocks of Alzheimer's Disease-related synaptotoxic species.Two NMR methodologies were developed together with this research: (1) A new approach to determine tyrosine sidechain pKa values in folded proteins; and(2) A simple and tunable way to speed up the NMR measurement of intrinsically disordered proteins using co-solute paramagnetic relaxation enhancement (PRE). The research presented in this dissertation can hopefully be beneficial not only for researchers involved in these subjects, but also to a wider NMR community.",

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